1. Field of the Invention
The present invention relates to the targeted delivery of liposomal complexes. In particular, the present invention relates to the reversible masking of liposomal complexes to allow for the systemic circulation of liposomal complexes, the efficient delivery of a nucleic acid or drug product to target tissues, and bypass non-target tissues/organs.
2. Description of the Related Art
The primary goal of gene therapy is to introduce genetic material, or a gene, into a given set of cells to provide those cells with a new protein manufacturing capacity. Success is measured by how well the added gene functions to produce the therapeutic protein. Successful gene therapy must overcome a number of problems, one of which is delivering the gene to the correct destination.
A “vector” is defined as the means for carrying foreign genetic material into a cell. Gene therapy vectors can be placed into two broad categories; the viral vectors and the non-viral vectors.
Viral vectors comprise about 90% of the currently used means of gene delivery. In viral vectors, the gene is engineered into a modified virus in hopes of capitalizing on the infectivity of the virus. Briefly, essential viral genes are removed to render the virus incapable of replicating or, in some cases, restricting viral replication to the cell targeted by the therapy. A therapeutic gene or genes replace the viral genes that have been removed. Over long spans of viral and host evolution, viral vectors have refined a wide range of gene-packaging and cell-entry mechanisms. Through highly specific means, typically involving cell surface receptors, these modified viruses gain entrance to cells. In the cell, they are able to evade intracellular degradation and to induce the expression of the virally introduced therapeutic genes. The principal viral vectors used are retroviruses, adenoviruses, adeno-associated viruses and herpes viruses.
Despite the extensive use of viral vectors, there are a number of disadvantages associated with the use of viral vectors. These disadvantages include: 1) the generation of immune responses to expressed viral proteins that subsequently kill the target cells required to produce the therapeutic gene product; 2) the random integration of some viral vectors into the host chromosome; 3) the clearance of viral vectors delivered systemically; 4) the difficulties in engineering viral envelopes or capsids to achieve specific delivery to cells other than those with natural tropism for the virus; 5) the potential recombination of the viral vector with DNA sequences in the host chromosome to generate a replication-competent, infectious virus; 6) the inability to administer certain viral vectors more than once; 7) the high costs of producing large amounts of high-titer viral stocks for use in clinical trials; and 8) the limited size of the nucleic acid that can be packaged in viral vectors.
Tremendous effort has been devoted to the development of non-viral delivery systems because of the perceived problems involved in using viral vectors. There are numerous patents that have described nucleic acid-containing liposomes. For example, the following U.S. Patents describe the use of liposomes to deliver nucleic acids: U.S. Pat. Nos. 6,316,024; 6,284,267; 6,271,206; 6,217,901; 6,159,745; 6,096,716; 6,056,973; 5,958,791; 5,891,468; 5,858,784; 5,820,873; 5,776,487; 5,756,353; 5,718,915; 5,662,930; 5,614,214; and 5,552,157.
Negatively charged, or pH-sensitive liposomes can be used. These liposomes entrap DNA rather than complex with it. Since both the DNA and the lipids used are negatively charged, repulsion occurs, although some DNA is entrapped in the aqueous interior of these liposomes.
The vast majority of liposomes used are cationic liposomes. Some liposomes are capable of enveloping anionic plasmid DNA and can carry and deliver naked DNA into targeted cells. Positively charged liposomal complexes can bind to the negatively charged cell surface and either be incorporated into cell membranes, liberating their DNA content into the cytoplasm, or be internalized in an endosome where the liposomes are ruptured and their contents released into the lysosome fused with the endosome. Therefore, cell entry through the endocytic pathway leads to the bulk of the DNA being degraded and a small amount released into the cell cytoplasm.
Liposomes have traditionally been considered biologically inert and can be standardized and regulated as drugs rather than as biologics. The use of liposomes in gene therapy provides multiple advantages: 1) the lack of immunogenicity; 2) the lack of clearance by complement using improved formulations; 3) the unlimited size of nucleic acids that can be delivered (from single nucleotides to large mammalian artificial chromosomes); 4) the ability to perform repeated administrations in vivo without adverse consequences; 5) the relative ease and lower cost in creating nucleic acid-liposomal complexes in large quantities for clinical trials; 6) the relative ease in creating targeted complexes for delivery and gene expression in specific cell types, organs or tissues; and 7) the increased patient safety due to few or no viral sequences present in plasmids used to deliver therapeutic genes, thus precluding the theoretical risk of oncogenesis or super-infection associated with the potential generation of an infectious virus.
The traditional disadvantage of non-viral delivery systems has been the low levels of delivery and gene expression produced by liposomal complexes in targeted tissues. One major problem encountered has been the first-pass elimination of cationic liposomal complexes prior to distribution to the rest of the body. Liposomal complexes administered into the systemic circulation by any route, excluding the intraarterial, are subject to first-pass clearance in the lung prior to their distribution elsewhere. Thus, the lung serves as a temporary clearing site for a number of agents, especially cationic compounds.
There exists a need for a method to systemically administer cationic liposomal complexes that will avoid first-pass clearance by the lung.